Exploring pharmaphylogeny from multiple perspectives: a case study on Lithospermeae

Lithospermeae Dumort., a tribe under the subfamily Boraginoidae, is a perennial herb containing approximately 470 species under 26 genera, primarily distributed in temperate and tropical regions. To gain a deeper understanding of the medicinal plants of Lithospermeae and better protect and develop plant medicinal resources, the phytochemistry, pharmacology, and traditional use of Lithospermeae with medicinal value were analyzed. Phylogenetic analysis was carried out based on the internal transcribed spacer sequence. Through spatial analysis and the species distribution model, the spatial distribution pattern of Lithospermeae medicinal plants was analyzed. Meanwhile, the relevant targets and pathways involved in the pharmacological effects of commonly used medicinal plants were predicted using network pharmacology to further explore the genetic origin of Lithospermeae and enrich the pharmaphylogeny of medicinal plants. In this study, the chemical composition, traditional efficacy, and modern pharmacological activity of Lithospermeae were collected for the first time and analyzed in combination with the geographical distribution model, molecular phylogeny, and network pharmacology. Based on our findings, the pharmaphylogeny of Lithospermeae was preliminarily discussed, providing the scientific basis for basic research regarding Lithospermeae. Concurrently, this study explored the relationship between the development of the regional medicinal plant industry and the protection of biodiversity. Furthermore, our findings provide direction and theoretical guidance for the study of the phylogenetic relationships in medicinal plants and the development of Lithospermeae medicinal plant resources.


Results
Plant systematics of Lithospermeae. In the phylogenetic tree, there are mainly four branches, with the first branch including Onosma and Maharanga DC. There are main notable morphological differences between Maharanga and Onosma. Maharanga has a ovoid-ellipsoid or obovoid corolla, contracted at both ends, abruptly expanding from a short tube into a relatively large inflated throat, with inflated ribs below lobes and deep furrows between them; throat unappendaged. In contrast, Onosma has a simple bell-shaped corolla, corolla blue, yellow, rarely white or red, tubular-campanulate or retrorse-conical, usually gradually expanded from the base upward, rarely abruptly expanded from the middle; throat unappendaged. Additionally, the base and the side of its anther are united 26 . Pollen morphology is a conservative characteristic of Onosma 26 . Figure 1 illustrates the morphology of some medicinal plants of Lithospermeae.
The second branch consists of Echium, Lobostemon, and Echium and begins as a cluster of panicles that gradually unites. It is obliquely funnel-shaped and is pubescent outside. The basal and lower leaves are blade lanceolate, the base is gradually narrowed into a handle, and long, rough hairs are present on both sides. Middle leaves. It is a corolla usually purplish-blue. There are two different classifications of Echium, viewing it either as an independent tribe of Boraginoideae or an entity under Lithospermeae. Evolution-wise, the pollen of Echium is at a higher level than the tricolpate pollen of Onosma. Based on the types of pollen pores, the authors believe that Echium has a classic tricolpate pollen, supporting the classification of Echium under Lithospermeae 27 . Lobostemon is characterized via a dry, papillary bilobed stigma and a hairless appearance 4 . The third branch consists of Alkanna, Arnebia, Buglossoides Moench, Glandora D.C.Thomas, Weigend & Hilger, Lithodora Griseb, Lithospermum, and Stenosolenium. Alkanna has basal leaves oblanceolate, eglandular white-tomentose with a dense indumentum of very short crisped hairs, and a yellow corolla. Lithodora has apparently stylar polymorphic since populations of all these species were found to have two morphs for style length: one morph with styles above the anthers (approach herkogamous) and the other positioned below (reverse herkogamous). Flowers are actinomorphic and sympetalous, form a floral tube, and have five small stamens inserted in the corolla tube. Lithospermum has short strigose, corolla actinomorphic, funnelform or salverform, throat with appendages or bands of hairs, or longitudinally crispate. Nutlets white or gray, ovoid, smooth, shiny or tuberculate; attachment scar at base adaxially. Buglossoides inflorescences are basically frondose-bracteose cincinni ("cymoids") with two to several flowers, usually distinctly elongating in fruit and with well-spaced, nutlet-bearing calyces. Its corolla may appear discoid and are in colors of white, blue, or light blue. Buglossoides is a small genus originally based on Lithospermum tenuiflorum and comprises seven species native to the Old World, mostly concentrated in the Mediterranean region. Glandora is a perennial shrub with flowers on top of leafy branches. Flowers are linear and petal-shaped, forming tubes. There are five small stamens in the corolla tube. Arnebia is an annual or perennial herb with bristly or pubescent. Flowers are often heterostylous. Corolla funnelform, commonly with hairs outside. Nutlets oblique-ovate, tuberculate, adaxially flat or slightly concave. The roots contain purple substances. (Fig. 2) Finally, Stenosolenium is a perennial herb with slender roots. Leaves are oblanceolate or lanceolate. Its corolla may appear purple, cyanish purple, or white. It has a slender tube and a hairy ring base. Figure 3 illustrates the phylogenetic tree of Lithospermeae species.
Geographical distribution. The medicinal plants of Lithospermeae are mainly distributed between 61° 14′ N and 14° 35′ N. Except for those in tropical rainforests, cold climates, tropical deserts, other climatic areas have distribution and abundant resources. The spatial distribution pattern illustrates high diversity in the Mediterranean climate regions, mainly including the Mediterranean coast, the Black Sea coast, southern Europe, and the northern African coast. High diversity is also observed in a few areas in western Asia, the western coast of southern Africa, and southwest and southeast coasts of Australia. The climate in these regions is usually hot and dry during the summer, and rainy in the winter. Notably, Lithospermeae species diversity is decreased at low latitudes. Alkanna is primarily distributed along the Mediterranean coast and the western coast of North America. Echium is mainly distributed along the Mediterranean coast and the western region of North America. Arnebia is primarily distributed in Africa and Asia. Lithospermum is mainly distributed in Asia. Onosma is mainly distributed along the Mediterranean coast and Tibet in China. Lithodora is primarily distributed near the Mediterranean Sea. Lobostemon is located in the southwest corner of Australia. Stenosolenium is distributed in eastern Asia. Figure 4A shows the distribution range of Lithospermeae. Moreover, the main distribution map of each genus is shown in the attachment (Supplementary Fig. S1-8).
From the perspective of suitable ecological zones, Alkanna is suitable for growing along the Mediterranean coast, central Asia, and western North America. Echium is suitable for growing along the Mediterranean coast, North America, southern South America, southwest Australia, and southeast Australia. Arnebia is suitable for growing in northern Africa and central Asia, along the Mediterranean coast. Lithospermum is suitable in eastern Asia and the Mediterranean coast, Europe, and North America. Onosma is more suitable on the Mediterranean coast. Lithodora is suitable for growing along the Mediterranean coast, along the western coast of North America, and the southeast corner of Australia. Lobostemon is suitable to grow in North America, Europe, and eastern Asia. Stenosolenium is suitable for growing in eastern Asia and parts of North America. Figure 4B  Chemical constituents. Research on the plant components of Lithospermeae mainly focuses on Arnebia, Echium, Lithospermum, and Onosma, followed by Alkanna, Lobostemon, and Stenosolenium. and Buglossoides, Lithodora, and Maharanga have been rarely reported 30,41,44 . The core components of Lithospermeae are purple naphthoquinones (e.g., shikonins, alkannins), pyrrolizidine alkaloids, and phenolic acids. The distribution of chemical constituents in each genus is summarized in Table 1. (The chemical structures file is attached. Supplementary Table S1). www.nature.com/scientificreports/ Traditional application and associated pharmacological activity. Traditionally, Lithospermeae medicinal plants are primarily used for burns, diarrhea, abscess, fever, detoxification, swelling, promoting blood circulation, dispelling wind pain, and contraception. Clinically, they are mainly used for their anti-inflammatory, antibacterial, antitumor, antioxidant, and wound-healing effects. The summary of its traditional application and pharmacological effects in individual genus distribution is shown in Table 2.
Network pharmacology study on representative genera of Lithospermeae. Screening  Obtaining disease targets. In Online Mendelian Inheritance in Man (OMIM) and GeneCards databases, disease targets were searched using the keywords "cancer", "inflammation", and "anxiety disorder". Targets with scores ≥ 5 were selected as potential targets. After merging the results of the two databases and deleting duplicates, 2976 cancer targets, 569 inflammatory targets, and 2102 anxiety disorder targets were obtained. Using the Venny 2.1.0 online platform, the intersection of component targets and disease targets was obtained. A total of 247 intersecting targets of Lithospermum and cancer, 98 intersecting targets of Arnebia and inflammation, and 156 intersecting targets of Echium and anxiety disorder were obtained.
KEGG pathway analysis and construction of "active ingredient-target-pathway" network. The Metascape database was used to analyze the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway of 247 core targets of the anticancer effects of Lithospermum, 98 core targets of the anti-inflammatory effects of Arnebia, and 156 core targets of the anti-anxiety effects of Echium. The first 20 most significant path-   www.nature.com/scientificreports/ and has an isohexenyl side chain, and the difference in its configuration comes from the chiral carbon atom on the side chain 29 . These compounds have good biological activity and are the main substances in the pharmacological effects of Lithospermeae. It has been well-established that the chemical constituents of plants are closely related to their efficacy and pharmacological activity. Different groups of medicinal plants and different parts of the same medicinal plant may have different varying traditional efficacy. The molecular phylogeny, geographical distribution, chemical composition, traditional application, and pharmacological effects of Lithospermeae were explored with respect to its pharmaphylogeny. Following the genetic theory, Lithospermeae plants are divided into three groups. Group one consists mainly of Onosma, followed by Maharanga, group two consists of Lobostemon and Echium, and group three consists of Alkanna, Arnebia, Stenosolenium, Lithodora, Lithospermum, Buglossoides, and Glandora.
Group 1: Onosma and Maharanga. While only a few studies on Maharanga, Maharanga has been used in traditional applications for constipation 17 , with antibacterial and antiviral pharmacological activities 30 . Onosma was mainly distributed on the Mediterranean coast and Tibet in China. The roots of Onosma were often reported to have been used as medicine for clearing heat and cooling blood, detoxification, treatment of rashes, and respiratory diseases 17 . The petals of Onosma were used for heart disease and rheumatic diseases. The main chemical constituents of Onosma are shown in Table 1, and its unique chemical components make its pharmacological www.nature.com/scientificreports/   www.nature.com/scientificreports/ activity remarkable. Onosma contains alkannins, shikonins, flavonoids, ferulic acids, and vanillic acids, which have been responsible for anti-inflammatory, wound healing, analgesic, and antibacterial activities (Tables 1, 2). Cypriots commonly used Onosma fruticosa for respiratory diseases 17 . The activity of the crude extract of Onosma bracteatum has shown promising results for asthma 31 . Group 2: Echium and Lobostemon. Echium is mainly distributed along the Mediterranean coast and in western North America. Echium is often used as whole herb. It can be seen from Table 2 that in the traditional application, it is used to treat rheumatic pain, abscess, respiratory tract infection, trauma, exterior relief, and diuresis. Particularly, its petals are used for antianxiety, sedation, and cold relief 17 . Table 1 depicts that the Echium primarily contains shikonins, phenolic acids, and pyrrolizidine alkaloids. Chemical components of Echium were notably associated with its traditional applications and pharmacological effects. Shikonins are often used for their antiinflammatory, antibacterial, antitumor, wound healing effects, and so on 32 . Phenolic acids have certain anticancer properties and are known to promote blood circulation 33 . The activity of the crude extract of Echium amoenum has been found to have anti-anxiety effects 34,35 , which was consistent with its traditional medicinal use. In previous studies, shikonin has been found to have a protective effect on the nervous system 36 , but the active ingredients responsible for its anti-anxiety effects are not clear. In this study, we used network pharmacology to analyze the molecular association between the chemical composition and anti-anxiety effects of Echium to explain its overall mechanism of action. In addition to shikonin, results of network pharmacology revealed the presence of caffeic acid, isobutyrylshikonin, and other compounds involved in the serotonergic pathway through targets such as monoamine oxidase B and APP. Compounds such as acetylshikonin and 2-methyl-n-butyrylshikonin regulate neuroactive ligand-receptor interaction pathways through targets such as recombinant adenosine a1 receptor (ADORA1) and adenosine a2a receptor (ADORA2A), allowing exertion of anti-anxiety effects (Fig. 5A,B).
Lobostemon is in the southwest corner of Australia and is mainly used for bacteriosis, ulcers, and wounds in traditional applications 17 . The chemical constituents of Lobostemon were mainly phenolic acids, flavonoids, and alkaloids. Clinically, it is mainly used for its anti-HIV and wound healing effects, etc. 24,37 . At present, there are only a limited number of reports on Lobostemon, and more studies are needed to better explore its pharmaphylogeny.
Part 1: Alkanna and Lithodora. Lithodora is mainly distributed on the Mediterranean coast and has been traditionally used for blood purification, chilblains, colds, cough, enteritis, fever, high blood pressure, and liver protection 17 . However, there are limited studies on its chemical constituents, with only aliphatic and ester compounds being reported (Table 1).
Alkanna is mainly distributed along the Mediterranean coast and the western coast of North America. In traditional applications, its roots are often used to treat diarrhea, abscess, wounds, burns, sore throat, etc. 17 . It mainly consists of quinone compounds, but phenolic acids, alkaloids, and flavonoids have also been found in Alkanna. Pharmacological studies have shown that in addition to analgesic, antibacterial, anticancer, antibacterial, anti-inflammatory, antioxidant, and wound healing effects, it also has benefits against nociceptive pain and cutaneous leishmaniasis ( Table 2).
Part 2: Buglossoides, Glandora, and Lithospermum. Lithospermum erythrorhizon is one of the commonly used Chinese herbal medicines 38 . Lithospermum diversity notably changed during the Middle Ages, with further dispersals of Lithospermum into North America, South America, and Africa observed 8 . In this study, it was found that Lithospermum was geographically limited to Asia. The results are inconsistent with previous reports, which may be due to the small sample size and the need to increase the sample information for further analysis. In addition to the common functions of Lithospermeae of clearing heat and detoxification, traditional applications for coughing, anti-inflammatory, promoting blood circulation, analgesia, and tonification for weakness have also been reported. It has been used as a diuretic and contraceptive in Europe and India, and its fruit is also known to assist in digestion 17 . Lithospermum mainly contains shikonins, alkannins, and pyrrolizidine alkaloids (Table 1). Lithospermum plants have antibacterial, anti-inflammatory, and antithrombotic effects, as well as the ability to palliate skin diseases (Table 2). Concurrently, crude extracts of Lithospermum erythrorhizon have been reported to assist in weight loss 39 . Lithospermum ruderale, mainly native to India, has also exhibited anti-gonadotropic activity 40 . From Fig. 1, Lithospermum canescens and Lithospermum ruderale can be seen to have similar sequences, but Lithospermum canescens has not been reported to have a contraceptive effect 17,40 . This highlight how genetic background is not the only determinant of the pharmacologic effects of medicinal plants. The potential of environmental factors in changing plant constituent's merits further study. In previous studies, shikonin and its derivatives were observed to inhibit the growth of tumor cells by activating miR-17-5p/ phosphatase and tensin homolog deleted on chromosome ten (PTEN)/protein kinase B (Akt), signal transducer and activator of transcription 3 (STAT3), phosphatidylinositol 3 kinase (PI3K)/Akt, and reactive oxygen species (ROS), among other pathways 32 . Through network pharmacology, we identified the signaling pathways closely related to the anticancer effect of Lithospermum. These pathways had the most enrichment targets, far more than others, and were mainly regulated via anhydroalkannin, arnebifuranone, and other compounds by regulating matix metalloproteinases1 (MMP1), recombinant cyclin dependent kinase 2 (CDK2), and epidermal growth factor 2 (EGF2). These findings provide a direction for Lithospermum as an antitumor drug (Fig. 6A,B).
Buglossoides and Glandora are relatively less studied. Buglossoides is produced in Asia, Europe, and North Africa. It is traditionally used for weight loss and has the biological activity of antioxidants 17 . Aliphatic and ester compounds have been identified in chemical studies. Glandora is native to North Africa and South Europe. It is worth noting that the synonym of Glandora diffusa in Glandora is Lithospermum diffusum and Lithodora diffusa 41,42 . In traditional applications, Glandora is used for its diuretic, depurative, and antihypertensive effects. It contains phenolic acids, flavonoids, fatty acids, etc., known to have a certain anticancer effect (Tables 1, 2). www.nature.com/scientificreports/ Part 2: Arnebia and Stenosolenium. Arnebia is mainly distributed in northern Africa and western Asia. Arnebia guttata and Arnebia euchroma are the source of authentic Chinese medicinal materials of 'Zicao' 38 . In traditional applications, it is often used for heat clearance, detoxification, burns, heart disease, skin diseases, blood circulation promotion, etc. 17 . Arnebia contains numerous quinone compounds, mainly shikonins and alkannins. It is commonly used for its anti-inflammatory, antibacterial, antioxidant, anticancer, and antithrombotic effects (Tables 1, 2). In addition to shikonin, polysaccharides in certain species have also been observed to exert antitumor effects. The crude extract has been found to inhibit the growth of human breast cancer cell line (MCF-7) cells and reduce the level of estrogen and progesterone in mice to achieve its anti-reproductive effect 43 . This seems to be consistent with the contraceptive effect of Lithospermum. The Arnebia species is often used for a variety of inflammatory conditions, such as arthritis, colitis, dermatitis, and so on 32 . Through network pharmacology, β-santalol, osthol, and arnebifuranone in species of Arnebia were identified to regulate the NOD-like receptor signaling pathway through recombinant mitogen activated protein kinase 8 (MAPK8), X-linked inhibitor of apoptosis protein (XIAP), mitogen-activated protein kinase 14 (MAPK14), and tumor necrosis factor (TNF) targets. Isovalerylshikonin, isovalerylalkannin, and arnebifuranone et al. regulate the MAPK signaling pathway through epidermal growth factor receptor (EGFR), MAPK8, and recombinant human protein kinase (AKT1) targets, exerting anti-inflammatory effects. Previous studies on the anti-inflammatory effect of Arnebia have identified MAPKs, TNF, and AKT as targets and MAPK as the signaling pathway. In this study, Arnebia was also found to regulate NOD-like receptor signaling pathways through XIAP targets. Although requiring further verification, this provides a new idea for harnessing the anti-inflammatory effects of Arnebia (Fig. 7A,B).
Stenosolenium is mainly distributed in eastern Asia. In traditional applications, the whole herb was used for dispelling wind, removing dampness, and managing joint pain. Meanwhile, the root has the effects of clearing heat, cooling blood, stopping bleeding, and relieving cough 17 . Stenosolenium contains quinones, phenolic acids, and pyrrolizidine alkaloids (Table 1). Further research is needed to better explore the pharmaphylogeny of Stenosolenium.
Alkanna, Lithospermum, Arnebia, Echium, and Onosma are the main sources of medicinal plants of Lithospermeae. Following palynology research, Echium and Onosma have a close genetic relationship. The chemical constituents of Echium and Onosma have similar traditional efficacy and pharmacological effects, suggesting their close relationship, which is consistent with the conclusion based on the ITS sequence. Lithospermum and Arnebia also have many similar characteristics as the main sources of medicinal 'Zicao' . For instance, they have similar efficacy in traditional applications (Table 2). In the current study, they were found to have a contraceptive effect. Their chemical constituents are mainly quinones, and their growth areas are similar, mainly in Asia. During the phylogenetic process, Lithospermum and Arnebia originate from the same evolutionary branch. Therefore, the consistency of plant efficacy between different groups suggests a subsequent relationship between the two in terms of chemical composition and phylogenetic development. This has important practical value for finding new drug sources by exploring the genetic relationship between plants.
At present, there are only a few studies on Lobostemon. However, following the surface characteristics of the stigma and the sinkage of the stem tip, Lobostemon has been reported to be similar to Echium 4 . Lithodora and Glandora are perennial shrubs with flowers on top of leafy branches. The flowers of the two genera are both linear and petal-shaped, forming tubes. They have five small stamens in the corolla tube. Glandora is a species that evolved from Lithodora 42,45 . Therefore, it is speculated that Lithodora and Glandora have a close genetic relationship. Buglossoides arvense originally belonged to the Lithospermum plant, and the ecological suitability and geographical locations of the two genera were also similar. Following the existing data, it is speculated that Buglossoides may have a close relationship with Lithospermum.
This study summarizes the chemical constituents, traditional applications, and pharmacological effects of Lithospermeae medicinal plants. Moreover, this paper reports on the genetic relationship among Lithospermeae through molecular phylogeny, species distribution model, and network pharmacology. At present, studies on Lithospermeae mainly focus on taxonomy and systematic position relationship, and preliminary studies on chemical constituents and pharmacological activities have demonstrated the rich diversity and activity potential of Lithospermeae. Our study explored the intrinsic correlation of the medicinal value of Lithospermeae. Based on previous pharmaphylogeny studies, we included a species distribution model and ecological adaptability evaluation to provide research basics for the future cultivation of Lithospermeae species. Meanwhile, network pharmacology was added to predict the targets and pathways of related diseases. Natural medicinal plants are gaining popularity among researchers due to their high efficacy, high quality, and low side effects. However, due to the growth environment and human factors, numerous medicinal plants have been damaged. Therefore, it is particularly urgent to find suitable substitutes and suitable cultivation techniques. This study not only enriches the pharmacy theory, helps to better understand, and tap the medicinal value of Lithospermeae, but also provides a reference for the development of other medicinal plants. Unfortunately, the medicinal value of 470 species of Lithospermeae plants has not attracted much attention and in-depth research for a long time, and only a few species have been highlighted for their chemical and pharmacological activities. We need a large number of studies on medicinal plants of the Lithospermeae to confirm our assumption. Moreover, the internal relationship among Lithospermeae medicinal plants remains unclear. These areas are worthy of further in-depth studies in the future. Phylogenetic studies mainly reflect the evolutionary relationship between species by constructing phylogenetic trees among species, to provide a basis for exploring the evolution of related organisms. Molecular systematics is the construction of a phylogenetic tree using deoxyribonucleic acid (DNA). It is believed that the